The long-term research goal of this investigator is to establish the mechanism by which a metabolic signal generated during glucose metabolism in pancreatic islets leads to stimulation of insulin secretion. The immediate focus of this proposal is to test specific hypotheses in yeast cells to determine their usefulness as a model to develop experimental approaches applicable to studies of glucose-induced insulin secretion in pancreatic islets where tissue availability for exploratory studies is quite limited. Glucose-induced insulin secretion in pancreatic islets, and glucose-induced cell proliferation in yeast, are both mediated by inositol lipid-derived intracellular messengers generated in response to metabolism of glucose. Since in both cases metabolism of the sugar is required, the mechanism is likely to be similar. Preliminary work with rat pancreatic islets supports the notion that a glycolytic intermediate (possibly, D- glyceraldehyde-3-phosphate) may be the specific metabolite which links the metabolism of glucose to stimulation of phospholipase C. The first aim of this proposal is to obtain additional evidence regarding the identity of this metabolite by examining the quantitative aspects of glucose-induced phosphoinositide response in yeast mutants defective in specific steps of the glycolytic pathway. Rationale is that mutants defective in a step prior to the key metabolite will not respond to glucose. The experimental approach will be to measure inositol 1,4,5-trisphosphate formation in [3H]-myoinositol labeled wild-type and mutant cells after exposure to glucose The second aim is to reproduce the phosphoinositide response to specific glucose metabolite(s) in isolated [3H]-myoinositol labeled plasma membrane preparations in the presence and absence of GTP and its analogues to ascertain the involvement of GTP-binding proteins in glucose-induced stimulation of the enzyme. A third related aim is to explore the possibility that the stimulation of phospholipase C by the specific glucose metabolite may involve inhibition of the GTPase activity of phospholipase C-specific GTP-binding protein. The experimental approach for this aim is to first obtain partially purified GTP-binding proteins by column-chromatography of solubilized plasma membrane proteins. Fractions containing the GTP-binding proteins will be identified by a [35S]GTPgammaS-binding assay. Effect of specific glucose metabolites on the GTPase activity will then be assayed by measuring the release of phosphate from added [gamma-32P]GTP in the presence and absence of specific -metabolites. The final aim is to evaluate the possibility that the cytoplasmic side of plasma membrane may possess a specific phospholipase C-coupled receptor which may bind a specific glucose metabolite. The experimental approach for this aim is to prepare radiolabeled ligands and then compare their binding to intact protoplasts and isolated plasma membrane in cold. It is anticipated that these studies will lead to development of experimental approaches to understand the mechanism of glucose-induced insulin secretion in pancreatic islets.